The papillomavirus life cycle is intimately linked with the differentiation state of the squamous epithelium as skin and the cervix. which it infects. One goal of this project is to set up an animal model that can be used to study the full viral life cycle. We The papillomaviruses are epitheliotropic viruses which induce benign and malignant lesions in a variety of squamous epithelia such are now ready to begin grafting human keratinocytes onto the backs of nude mice. In this environment these cells form a fully differentiated squamous epithelium. Wild type and mutant human papillomaviruses will be introduced into these cells to study the roles of cis elements and trans factors in the viral life cycle. Previously we have used in situ hybridization to demonstrate that alternative splicing of BPV-1 late pre-mRNAs is regulated in a differentiation dependent manner. Several cis-elements have been identified which regulate splice site choice. Immediately downstream of the first of two alternative 3' splice sites is a purine-rich positive element known as an exonic splicing enhancer (ESE) which is required for efficient utilization of that splice site. Immediately downstream of this element is a pyrimidine-rich negative element known as an exonic splicing suppressor (ESS). Together these two elements form a bipartite splicing regulatory element which modulates utilization of the upstream splice site. Only two other bipartite splicing regulatory elements are known. Further studies on the ESS indicate that it can suppress splicing of HIV-1 and RSV pre-mRNAs which contain suboptimal splice sites but has no effect on a -globin pre-mRNA containing strong splice sites. A third element similar to the exonic splicing enhancer has been identified a short distance upstream of the second alternative 3' splice site. Although this element can function as a splicing enhancer when located near a 3' splice site in an exonic position, we speculate that it functions in its normal location as an intronic splicing suppressor. This arrangement potentially allows the coordinated regulation of two alternative splice sites by the same trans-acting factors. Combined UV crosslinking and immunoprecipitation experiments indicated that both splicing enhancers bind a subset of the SR family of splicing factors (ASF/SF2, SRp55, and SRp75). Mutational analysis indicated that this binding is functionally relevant. The ESS binds a 65 kDa protein which may be the splicing factor U2AF65. We are currently investigating whether the activity of these factors is regulated by differentiation of the epithelial cells. In other studies we have screened an expression library for proteins which bind to the BPV-1 early 3' untranslated region. Only one protein, a bovine Y-box protein, was identified. The functional significance of this binding is still unknown.